Chemometrics-based evaluation on effect of sonication, contact time and solvent-to-solid ratio on total phenolics and flavonoids, fatty acids and antibacterial potency of Carica papaya seed against S. enteritidis, B. cereus, V. vulnificus and P. mirabilis

This study was aimed on extraction optimization of antibacterial agent from Carica papaya seed against S. enteritidis, B. cereus, V. vulnificus and P. mirabilis as affected by sonicationassisted extraction (SAE), contact time (CT) and solid-to-solvent ratio (SSR). The principal component analysis (PCA) and individual evaluation approaches identified that no SAE, 8 CT and 1:10 SSR was the best treatment with the highest antibacterial potency. The PCA identified no SAE, 8 CT, and 1:5 SSR as the second-beat treatment. The yield, total phenolic compound (TPC), C18:1n9t and C16:1 fatty acids (FAs) in no SAE, 8 CT and 1:10 SSR treatment inhibited the growth of B. cereus, V. vulnificus and P. mirabilis while C21:0 and C15:0 in 30 min SAE, 8 CT and 1:2 SSR inhibited the S. enteritidis growth. The yield, TPC, C18:1n9t and C16:1 FAs, and C6:0 and C24:1n9, C20:1, C4:0 and C20:0 FAs had antagonistic effects on B. cereus, V. vulnificus and P. mirabilis growths. The C21:0, C15:0, . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ; https://doi.org/10.1101/2022.02.10.479904 doi: bioRxiv preprint

Generally, direct extraction without manipulating the extraction treatments is applied to study the antibacterial properties of natural by-products. Recently, new techniques such as microwave-assisted extraction (Mayer et al., 2008) and supercritical CO 2 extraction (Zhao & Zhang, 2013) have been utilised to extract antibacterial compounds. However, relative to those techniques, the use of sonication-assisted extraction (SAE) is more convenient, affordable, environmentally friendly and industrially employed in local companies, by which the active compounds could be extracted in a shorter time and higher efficiency (Ya-Qin Ma et al., 2008). Time of contact (CT) is a necessary treatment to be optimised to minimise the process's energy cost (Spigno et al., 2007). Longer contact time exposed active sites of the solid area, improved sample homogeneity (Chinn et al., 2011) and increased extraction yield (Romdhane & Gourdon, 2002). Solid-to-solvent ratio (SSR) was also reported to be a significant variation in the extraction of natural by-products (Pinelo et al., 2005) as it allowed maximum surface contact between solid and solvent (Zhang et al., 2007) and increased mass transfer rate (Pinelo et al., 2005). Both treatments also affected phenolic Wong, Tan, & Ho (2013) and flavonoid contents (Tan et al., 2011). Studies on contact time affecting antioxidative properties for fruit by-products such as citrus peel (Y.-Q. Ma et al., 2009) and SSR on grape seed (Pinelo et al., 2005) have been reported, but not for antibacterial properties.
A common approach to optimise the extraction of antibacterial compounds is by carrying out extraction treatment and evaluating the treatment effect individually, where only minimum inhibitory concentration (MIC) was considered. This approach brings minimal information for researchers to investigate the antibacterial activity of Carica papaya seed extract. Sani et al. (2017a) proposed including total phenolic compound and flavonoid compounds since these phytochemicals were reported to render antibacterial activities (Alonso-Esteban et al., 2019). However, to gain more information on the variables contributing to the antibacterial potency, fatty acids were included since 80.23% of Carica papaya seeds are dominated by the fatty acids and their esters . Therefore, our study quantified 37 fatty acids in their ester form and evaluated the effect of treatments and these variables using chemometric-based approachs i.e., principal component analysis (PCA) suitable for a multivariate dataset (Sani et al., 2021b). Very negligible report employed the chemometricbased approach to evaluate the antibacterial potency, especially on Carica papaya seed extract to date. It is also recommended to add MIC 50 and MIC 0 as additional antibacterial variables since these varaiables have variation that could render a more meaningful insight in the PCA results. It is anticipated that this study could provide a new insight about the antibacterial activities of Carica papaya seed extract, promote the application of cheap and ubiquitous by-products, and provide economic extraction procedures. This study will indirectly accelerate the quest for natural antibacterials.

Experimental design
The experiment was designed as depicted in Figure 1. The experiment was divided into three effects which involved the effect of sonication-assisted extraction (SAE), contact time (CT) and solid-to-solvent ratio (SSR). The best treatment from each effect was applied in the following effect. For instance, the best treatment for the SAE effect was used in the CT effect, and both the best treatment from SAE and CT effect was then employed in the SSR . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 effect. For each effect, the yield, total phenolic content (TPC) and total flavonoid content (TFC) of the extract ware determined as well as the antibacterial activities, consisting of minimum inhibitory concentration (MIC), MIC 50 , and MIC 0 of S. enteritidis, B. cereus, V. vulnificus and P. mirabilis. Evaluation on the (1) individual treatment and variable and (2) treatments and multivariable via chemometrics were carried out and compared. The correlations among the variables were assessed, and the variables that characterised the treatment were identified.

Extraction of phytochemicals
Methanol (MeOH) was used as solvents in the extraction, according to Sani et al. (2020) (Switzerland) before (T 0 ) and after (T 24 ) incubation. A TSB medium incubated with a target bacterium (without an antibacterial agent) was used as a positive control of growth in the twelfth well in each row.
The MIC was defined as the lowest concentration of antibacterial agent showing a complete growth inhibition of the tested bacterial strains, which was related to a difference absorbance of T 24 and T 0 (T 24 -T 0 ) equal to zero or negative values.
The graphs of percentage inhibition for each extraction treatment were plotted, and the MIC was compared to the percentage inhibition where MIC had 100% bacterial inhibition. MIC 50 was determined by calculating the concentration that gave 50% inhibition by using linear regression (y = mx + c), where y = 50%, m = slope of regression, c = intercept of regression and x = concentration of extract at 50% inhibition. From the percentage inhibition graphs, the . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ; https://doi.org/10.1101/2022.02.10.479904 doi: bioRxiv preprint concentration of extract which gave 0% inhibition was also determined as MIC 0 . All determinations were performed in triplicate (Sowhini et al., 2020).

Quantitation of total phenolic content (TPC)
Total phenolic contents of Carica papaya crude extracts were determined by colourimetry assay with Folin-Ciocalteu according to Sani, Bakar, Rahman, & Abas (2017a). An amount of 0.05 g Carica papaya seed extract was diluted to 100 mL in a volumetric flask, and 1 mL of the diluted extract was mixed with 1 mL of 1:10 diluted Folin-Ciocalteu reagent (Sigma-Aldrich, Switzerland) in a 5 mL volumetric flask wrapped with aluminium foil and vortexed for 10 s. The mixture was then incubated at 30 o C for 5 min, mixed with 1 mL sodium carbonate (10%, w/v) solution (Sigma-Aldrich, Switzerland) and marked up to the volume.
Then, the mixture was vortexed (VTX-3000L, Copens Scientific, Germany) for another 10 s and incubated in the dark at 30 o C for 30 min. The mixture produced a blue aqueous layer, and its absorbance was measured using a spectrophotometer (U-2810 Hitachi, Japan) at 747 nm against methanol as a blank in triplications. The exact incubation procedure was employed to prepare 0-10 mg/L gallic acid standard (Sigma-Aldrich, Switzerland) solutions in methanol.
A calibration curve of gallic acid absorbance versus concentration was plotted, and the concentration of Carica papaya seed extract was computed from the calibration equation.
Results were expressed as gallic acid equivalent (GAE) in mg/g of dry weight (DW) of the sample (mg GAE/g DW).
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Quantitation of total flavonoid content (TFC)
A series of quercetin working standards at 0 -12.5 mg/L, including ethanol as blank, was measured spectrophotometrically against ethanol at 438 nm, and a calibration curve was established. Total flavonoid contents of Carica papaya crude extracts were determined following Sani et al. (2017b). An amount of 0.05 g Carica papaya seed extract was diluted in a 100 mL volumetric flask with ethanol. A volume of 1.25 mL the diluted extract was mixed

Analysis of fatty acids methyl esters by gas chromatography-mass spectrometer
A series of working FAMEs standard in hexane ranging from 0.0005 -3 mg/mL was prepared in 1 mL volumetric flask and injected into gas chromatography-mass spectrometry (GC/MS). A concentration of 0.01 g/mL Carica papaya seed extract was mixed with 0.6 mL of hexane and 0.4 mL of 1 M solution of sodium methoxide. Then, the mixture was vortexed for 30 s. A volume of 0.6 mL of top hexane layer was analysed by GC/MS for FAME quantification.
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Separation and detection of FAMEs was carried out on an Agilent-Technologies 7890A gas chromatography (GC) system equipped with an Agilent-Technologies 5975 mass spectrometer (MS) system (Agilent Technologies, USA). The working standards and top hexane layer of the Carica papaya seed extracts were injected into an injector temperature maintained at 260°C. A volume of 1 µL of the standard and extracts was split at 1:10 ratio and eluted into the GC system by helium at 1 mL/min flow rate. The FAMEs were separated by an HP-88 capillary column (100 m x 0.25 mm, film thickness 0.20 µm) with an oven temperature program at (1) 150°C for 5 min, (2) heated to 240°C at 4°C/min and (3)  . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ;

Statistical analysis
Data were expressed as mean ± standard deviation of extraction yield, TPC and TFC and MIC 50 . One-way analysis of variance (ANOVA) with Tukey's test was conducted using XLSTAT-Pro (2014) statistical software (Addinsoft, Paris, France) to determine the significant difference between the means at 95% confidence level (p < 0.05) for extraction yield, TPC and TFC and MIC 50. In this study, a principal component analysis ( Based on these FLs, the variable correlations and their contributions to extraction treatments were assessed. . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ;

MIC, MIC 50 and MIC 0 as affected by sonication, contact time and the solid-tosolvent ratio of Carica papaya seed extract
The MIC test, a descriptive antibacterial method, had only given limited information on bacterial inhibition (Vigil et al., 2005) and inadequate comparison between extraction treatments. Thus, Patton et al. (2006) (Table 1). However, as we performed the significant test on these data, the MIC 50 from these treatments were not significantly different from the no SAE treatment. The no SAE treatment gave the lowest MIC 0 than SAE treatments for all tested pathogens (Table 1).
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (Chinn et al., 2011). The MIC of these microorganisms also exhibited a strong correlation with contact time (Table 1) . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made where the MIC 0 for these microorganisms remained at < 0.02 mg/mL. The MIC 0 from 1:10 SSR also had presented the lowest value for all microorganisms (< 0.02 mg/mL). The 1:10 SSR had lowered the MIC 0 of S. enteritidis to < 0.02 mg/mL from 0.70 mg/mL of 1:2 SSR ( . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ;  (2010). This occurrence was possibly due to the extraction reaching equilibrium before 15 min SAE contact time (Tian et al., 2013), reducing solvent's permeability into cell structures on account of insoluble lipids existence on the ruptured cell (Tian et al., 2013) or re-adsorption of active components because of a large specific area of the ruptured cells (Dong et al., 2010). The extraction yield of Carica papaya seed extract also increased as contact time increased ( . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The 8 h CT also exhibited the highest amount of TPC (20.10 mg/g) with a significant difference (p < 0.05) value as compared to other treatments due to longer contact time had improved surface area and slurry homogeneity of the sample; hence, provided a positive influence on phenolics (Chinn et al., 2011) by allowing the progressive release of phenolics from solid matrix to solvent (Spigno et al., 2007). Thus, the 8 h CT was the best CT treatment (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ; https://doi.org/10.1101/2022.02.10.479904 doi: bioRxiv preprint difference (p < 0.05) of TFC (2.31 -4.94 mg QE/g DW) in Table 2 as compared to no SAE treatment TFC (1.91 mg QE/g DW), where the 60 min SAE demonstrated the highest value, possibly due to flavonoids in the Carica papaya seed were in the form of flavonoid glycosides, which have thermal stability (Biesaga & Pyrzyńska, 2013;Ya Qin Ma et al., 2008). Since the Carica papaya seed used in this study did not undergo an acid hydrolysis process, only flavonoid glycosides were extracted out (Khoddami et al., 2013). Pan, Yu, Zhu, & Qiao (2012) claimed that the highest recovery of TFC in hawthorn seed was obtained at 91 o C, which exceeded the temperature observed in this study (70°C), indicating the increment of temperature during SAE had not destabilised flavonoid glycosides. The increment of temperature during SAE was also reported to enhance solubility, increase the diffusion coefficient, and increase the extraction rate of TFC (Cacace & Mazza, 2003). From this finding, the no SAE was the best treatment compared to other SAE treatments.
On the other hand, the CT effect on TFC exhibited an inverse trend, where 2 h CT yielded the highest (4.43 mg/g) with a significant amount (p < 0.05). Even though Spigno et al. (2007) found that the optimum contact time for flavonoids in grape marc extraction was 5 h, our result showed the highest TPC, which was in line with the TPC of Salvia officinalis by Durling et al. (2007) at 8 h.
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Correlation of total phenolic and flavonoids, fatty acids and antibacterial potency of Carica papaya seed extract on the extraction treatments
The purposes of using PCA in this study were to describe the correlation and distribution of yield, TPC, TFC and fatty acids on the antibacterial potency of Carica papaya seed extracts as affected by the SAE, CT and SSR. Table 3 shows the characteristics of the analytical curves with the R 2 values. The R 2 > 0.98 indicated that the analytical curve values had established linear regression models, which were adequate for the FAMEs determination in the Carica papaya seed extract (Sani et al., 2021b). By analysing the FAMEs that take about 80.23% of the Carica papaya seed extract , we could investigate their influences on the yield, TPC and TFC on the pathogens inhibition via a chemometric technique such as PCA.
The PCA exhibited two principal components (PCs) entailing 36 variables that represent cumulative variability of 46% with an eigenvalue (EV) of 6.57 in Figure 2  . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ;  FAMEs were dominant in Carica papaya seed extract, other compounds such as organic acids, fatty aldehydes, sterols, nitriles and amides that had a phenolic backbone may render the antibacterial potency ; therefore, denoted the high TPC in this study.

90
The yield, TPC, C18:1n9t and C16:1 also had a negative correlation with the antibacterial variables except for MICSE on the opposite side of Figure 1  mirabilis. Likewise, since the C6:0 and C24:1n9 were located in the same quadrant of these antibacterial variables, they were moderately rendered increment growth of the pathogens.
The opposite direction of the yield, TPC, C18:1n9t and C16:1 against C6:0 and C24:1n9, C20:1, C4:0 and C20:0 also indicated that these variables had antagonistic effects on the potency of Carica papaya seed extract. Hence, partially purified Carica papaya seed extract or a mixture of phenolics and purified C18:1n9t and C16:1 shall be employed instead of the crude extract to enhance the antibacterial potency of the Carica papaya seed extract against B.cereus, V.vulnificus, and P. mirabilis. This was evident since organic acids and sterols from Carica papaya seed extract have rendered MIC at 2.81 mg/mL, 0.35 mg/mL, and 1.41 mg/mL towards B. cereus, V.vulnificus, and P. mirabilis, respectively (Sani et al., 2021a). An interesting observation showed that C15:0, C18:0, C16:0, C14:0, C18:2n6c, C18:1n9c, C23:0 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 and C20:2 had no effect on the inhibition of B.cereus, V.vulnificus and P. mirabilis. This finding was due to their 90• direction towards the MIC, MIC 50 and MIC 0 of these pathogens.
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ;  . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ; https://doi.org/10. 1101/2022 Further investigation on the antibacterial variables, only MIC 50 SE, MIC 50 VV and MIC 0 SE had the strong FL (Figure 2 (a)) due to PCA measures variances in the dataset. This variance measurement fit for continuous variables compared to the end-point MIC value of pathogens (Sowhini et al., 2020). Hence, recording the antibacterial activity of Carica papaya seed extract would be better in MIC 50 and MIC 0 instead of MIC.
The biplot in Figure 2 (b) exhibited four specific clusters, i.e., 1:10 SSR, 1:5 SSR, 30 min SAE and 15 min SAE, and two mixed clusters. The 1:10 SSR was positioned at the most left of the F1 axes, followed by the 1:5 SSR where the yield, TPC, C18:1n9t and C16:1 were high while C6:0, C24:1n9 and C20:1 were low in these clusters. The extraction of Carica papaya seed through no SAE, 8 h CT and 1:10 SSR would be the best treatments to exert the highest antibacterial potency because of their opposite position of the antibacterial variables except MICSE. Likewise, the no SAE, 8 h CT and 1:5 SSR extraction treatments could be the second-best option.
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ; https://doi.org/10. 1101/2022 The 30 min SAE cluster was also dominant as it is located to the left side of the F2 axes in Figure 2 (b). High C21:0 and C15:0 and low C20:2 and C23:0 characterised this cluster which also denoted that the extraction treatments 30 min SAE, 8 h CT and 1:2 SSR could inhibit the S. enteritidis as depicted with the opposite position of this cluster against the MICSE.
Nevertheless, the 15 min SAE cluster was positioned with the antibacterial variables except for MICSE, indicating 15 min SAE, 8 CT, and 1:2 SSR could not enhance the antibacterial potency of Carica papaya seed extract (Figure 2 (b)). This was due to high C6:0, C24:1n9 and C20:1 and low yield, TPC, C18:1n9t, and C16:1 may render antagonistic effects and had facilitated the pathogens' growth. One mixed cluster consisting of 1h SAE, 2 h CT and 4 h CT also had indicated they could not inhibit the pathogens' growth. In the same position, the C4:0 and C20:0 were dominant in this cluster. Another mixed cluster had the extracts produced from the standard extraction treatments, i.e., no SAE, 8 h CT and 1:2 SSR. The C23:0, C20:2, and C11:0 were high in this cluster, but they could not inhibit the S. enteritidis.
To note, the two mixed clusters and 15 min SAE cluster were located at the centre of the F1 and F2 axes; therefore, they were the least significant extraction treatment in this study.
From these evaluations, the no SAE, 8 h CT and 1:10 SSR would be the best treatments to inhibit the B.cereus, V.vulnificus, and P. mirabilis growths while 30 min SAE, 8 h CT and 1:2 SSR could be the best extraction treatments to inhibit the S. enteritidis. It is also recommended to apply PCA to facilitate the interpretation of multivariable dataset instead of evaluating individual variables (Sani et al., 2021b). Besides providing information on the best extraction treatments, the PCA presents in-depth information on the variable correlations and . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ; https://doi.org/10. 1101/2022 proposes variables with synergistic and antagonistic effects that the evaluation of individual variables could not provide.

Conclusion
The chemometrics-based evaluation via PCA was superior over the individual evaluation treatment on antibacterial potency of Carica papaya seed extract. The no SAE, 8 h CT and 1:10 SSR were the most efficient treatments for extracting antibacterial compounds from Carica papaya seed than other SAE and CT treatments due to the highest yield and TPC.
These treatments also gave the lowest MIC, MIC 50 and MIC 0 of S. enteritidis, B. cereus, V. vulnificus and P. mirabilis. Only B. cereus and P. mirabilis were sensitive against no SAE, 8 h CT and 1:10 SSR. All tested pathogens were sensitive against 1:10 SSR. Besides identifying the best treatment, the PCA had successfully identified the characterising variables on the treatments and synergistic and antagonistic effects of the variables. Although the PCA could delineate this information, only 46% of the dataset was explained in this study; hence, more variables and samples will be included in future research. The results and methods described here could help optimise extraction procedures through antibacterial test assessment and thus help reveal novel antibacterial compounds, which are expected to serve as an antibacterial agent food preservative. a . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 10, 2022. ; https://doi.org/10.1101/2022.02.10.479904 doi: bioRxiv preprint

Conflict of interest statement
We declare no conflict of interest.

Research involving human participants and/or animals
We declare no human and/or animals involved in this study.
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